Costimulatory Chimeric Antigen Receptor T Cells Targeting IL13Rx2

ABSTRACT

Chimeric transmembrane immunoreceptors (CAR) which include an extracellular domain that includes IL-13 or a variant thereof that binds interleukin-13Rα2 (IL13Rα2), a transmembrane region, a costimulatory domain and an intracellular signaling domain are described.

CLAIM OF PRIORITY

This application claims priority under 35 U.S.C. §365(c) toInternational Patent Application PCT/US2015/051089, filed on Sep. 18,2015, which claims priority under 35 U.S.C. §119(e) to provisional U.S.Patent Application 62/053,068, filed on Sep. 19, 2014, the entirecontents of each which are hereby incorporated by reference.

BACKGROUND

Tumor-specific T cell based immunotherapies, including therapiesemploying engineered T cells, have been investigated for anti-tumortreatment. In some cases the T cells used in such therapies do notremain active in vivo for a long enough period. In some cases, thetumor-specificity of the T cells is relatively low. Therefore, there isa need in the art for tumor-specific cancer therapies with longer termanti-tumor functioning.

Malignant gliomas (MG), which include anaplastic astrocytoma (AA-gradeIII) and glioblastoma (GBM-grade IV), have an incidence rate ofapproximately 20,000 new cases diagnosed annually in the United States.According to the American Brain Tumor Association total prevalence ofindividuals living with a malignant brain tumor, based on United States2010 census data, is roughly 140,000 persons. Although MG is a raredisease, it is highly aggressive and heterogeneous with respect to itsmalignant behavior and nearly uniformly lethal. Current standard-of-caretherapies for high-grade MG yield only short term benefits, and thesebrain tumors are virtually incurable. Indeed, even with modern surgicaland radiotherapeutic techniques, which often exacerbate the alreadysevere morbidities imposed by location in the central nervous system(CNS), the 5-year survival rates are quite low. Furthermore, for themajority of patients who relapse with disease, there are few therapeuticoptions. Thus, there is a significant need for more effective therapies,particularly for those patients that have recurred/progressed followingfrontline therapies, and participation of this patient population inclinical trials is warranted.

Adoptive T cell therapy (ACT) utilizing chimeric antigen receptor (CAR)engineered T cells may provide a safe and effective way to reducerecurrence rates of MG, since CAR T cells can be engineered tospecifically recognize antigenically-distinct tumor populations(Cartellieri et al. 2010 J Biomed Biotechnol 2010: 956304; Ahmed et al.2010 Clin Cancer Res 16:474; Sampson et al. 2014 Clin Cancer Res 20:972;Brown et al. 2013 Clin Cancer Res 2012 18:2199; Chow et al. 2013 MolTher 21:629), and T cells can migrate through the brain parenchyma totarget and kill infiltrative malignant cells (Hong et al. 2010 ClinCancer Res 16:4892; Brown et al. 2007 J Immunol 179:3332; Hong et al.2010 Clin Cancer Res 16:4892; Yaghoubi 2009 Nat Clin PRact Oncol 6:53).Preclinical studies have demonstrated that IL13Rα2-targeting CAR+ Tcells exhibit potent major histocompatibility complex (MHC)-independent,IL13Rα2-specific cytolytic activity against both stem-like anddifferentiated glioma cells, and induce regression of established gliomaxenografts in vivo (Kahlon et al. 2004 Cancer Res 64:9160; Brown et al.2012 Clin Cancer Res 18:2199).

SUMMARY

Described herein are chimeric transmembrane immunoreceptors (chimericantigen receptors or “CARs”) which comprise an extracellular domain, atransmembrane region and an intracellular signaling domain. Theextracellular domain is made up of an IL-13 ligand that bindsinterleukin-13Rα2 (IL13Rα2) and, optionally, a spacer, comprising, forexample a portion human Fc domain. The transmembrane portion includes aCD4 transmembrane domain, a CD8 transmembrane domain, a CD28transmembrane domain, a CD3 transmembrane domain or a 4IBB transmembranedomain. The intracellular signaling domain includes the signaling domainfrom the zeta chain of the human CD3 complex (CD3ζ) and one or morecostimulatory domains, e.g., a 4-1BB costimulatory domain. Theextracellular domain enables the CAR, when expressed on the surface of aT cell, to direct T cell activity to those cells expressing IL13Rα2, areceptor expressed on the surface of tumor cells, including glioma.Importantly, the IL13Rα2 binding portion of the CAR includes an aminoacid modification, such as an E13Y mutation, that increases bindingspecificity. The inclusion of a costimulatory domain, such as the 4-1BB(CD137) costimulatory domain in series with CD3 in the intracellularregion enables the T cell to receive co-stimulatory signals. T cells,for example, patient-specific, autologous T cells can be engineered toexpress the CARs described herein and the engineered cells can beexpanded and used in ACT. Various T cell subsets can be used. Inaddition, the CAR can be expressed in other immune cells such as NKcells. Where a patient is treated with an immune cell expressing a CARdescribed herein the cell can be an autologous or allogenic T cell. Insome cases the cells used are CD4+ and CD8+ central memory T cells(T_(CM)), which are CD45RO+CD62L+, and the use of such cells can improvelong-term persistence of the cells after adoptive transfer compared tothe use of other types of patient-specific T cells.

Described herein is a nucleic acid molecule encoding a chimeric antigenreceptor (CAR)r, wherein the chimeric antigen receptor comprises: humanIL-13 or a variant thereof having 1-10 (e.g., 1 or 2) amino acidmodifications; a transmembrane domain selected from: a CD4 transmembranedomain or variant thereof having 1-10 (e.g., 1 or 2) amino acidmodifications, a CD8 transmembrane domain or variant thereof having 1-10(e.g., 1 or 2) amino acid modifications, a CD28 transmembrane domain ora variant thereof having 1-10 (e.g., 1 or 2) amino acid modifications,and a CD3ζ transmembrane domain or a variant thereof having 1-10 (e.g.,1 or 2) amino acid modifications; a costimulatory domain; and CD3ζsignaling domain of a variant thereof having 1-10 (e.g., 1 or 2) aminoacid modifications.

In various embodiments the costimulatory domain is selected from thegroup consisting of: a CD28 costimulatory domain or a variant thereofhaving 1-10 (e.g., 1 or 2) amino acid modifications, a 4-IBBcostimulatory domain or a variant thereof having 1-10 (e.g., 1 or 2)amino acid modifications and an OX40 costimulatory domain or a variantthereof having 1-10 (e.g., 1 or 2) amino acid modifications. In certainembodiments, a 4IBB costimulatory domain or a variant thereof having1-10 (e.g., 1 or 2) amino acid modifications in present.

Additional embodiment the CAR comprises: a variant of a human IL13having 1-10 amino acid modification that increase binding specificityfor IL13Rα2 versus IL13Rα1; the human IL-13 or variant thereof is anIL-13 variant comprising the amino acid sequence of SEQ ID NO:3 with 1to 5 amino acid modifications, provided that the amino acid at position11 of SEQ ID NO:3 other than E; two different costimulatory domainsselected from the group consisting of: a CD28 costimulatory domain or avariant thereof having 1-10 (e.g., 1 or 2) amino acid modifications, a4IBB costimulatory domain or a variant thereof having 1-10 (e.g., 1 or2) amino acid modifications and an OX40 costimulatory domain or avariant thereof having 1-10 (e.g., 1 or 2) amino acid modifications; twodifferent costimulatory domains selected from the group consisting of: aCD28 costimulatory domain or a variant thereof having 1-2 amino acidmodifications, a 4IBB costimulatory domain or a variant thereof having1-2 amino acid modifications and an OX40 costimulatory domain or avariant thereof having 1-2 amino acid modifications; human IL-13 or avariant thereof having 1-2 amino acid modifications; a transmembranedomain selected from: a CD4 transmembrane domain or variant thereofhaving 1-2 amino acid modifications, a CD8 transmembrane domain orvariant thereof having 1-2 amino acid modifications, a CD28transmembrane domain or a variant thereof having 1-2 amino acidmodifications, and a CD3ζ transmembrane domain or a variant thereofhaving 1-2 amino acid modifications; a costimulatory domain; and CD3ζsignaling domain of a variant thereof having 1-2 amino acidmodifications; a spacer region located between the IL-13 or variantthereof and the transmembrane domain (e.g., the spacer region comprisesan amino acid sequence selected from the group consisting of SEQ ID NO:4, 14-20, 50 and 52); the spacer comprises an IgG hinge region; thespacer region comprises 10-150 amino acids; the 4-1BB signaling domaincomprises the amino acid sequence of SEQ ID NO:6; the CD3ζ signalingdomain comprises the amino acid sequence of SEQ ID NO:7; and a linker of3 to 15 amino acids that is located between the costimulatory domain andthe CD3ζ signaling domain or variant thereof. In certain embodimentswhere there are two costimulatory domains, one is an 4-IBB costimulatorydomain and the other a costimulatory domain selected from: CD28 andCD28gg

In some embodiments: nucleic acid molecule expresses a polypeptidecomprising an amino acid sequence selected from SEQ ID NOs: 10, 31-48and 52; the chimeric antigen receptor comprises a IL-13/IgG4/CD4t/41-BBregion comprising the amino acid of SEQ ID NO:11 and a CD3ζ signalingdomain comprising the amino acid sequence of SEQ ID NO:7; and thechimeric antigen receptor comprises the amino acid sequence of SEQ IDNOs: 10, 31-48 and 52.

Also disclosed is a population of human T cells transduced by a vectorcomprising an expression cassette encoding a chimeric antigen receptor,wherein chimeric antigen receptor comprises: human IL-13 or a variantthereof having 1-10 amino acid modifications; a transmembrane domainselected from: a CD4 transmembrane domain or variant thereof having 1-10amino acid modifications, a CD8 transmembrane domain or variant thereofhaving 1-10 amino acid modifications, a CD28 transmembrane domain or avariant thereof having 1-10 amino acid modifications, and a CD3ζtransmembrane domain or a variant thereof having 1-10 amino acidmodifications; a costimulatory domain; and CD3ζ signaling domain of avariant thereof having 1-10 amino acid modifications. In variousembodiments: the population of human T cells comprise a vectorexpressing a chimeric antigen receptor comprising an amino acid sequenceselected from SEQ ID NOs: 10, 31-48 and 52; the population of human Tcells are comprises of central memory T cells (Tcm cells) (e.g., atleast 20%, 30%, 40%, 50% 60%, 70%, 80% of the cells are Tcm cells; atleast 15%, 20%, 25%, 30%, 35% of the Tcm cells are CD4+ and at least15%, 20%, 25%, 30%, 35% of the Tcm cells are CD8+ cells).

Also described is a method of treating cancer in a patient comprisingadministering a population of autologous or allogeneic human T cells(e.g., autologous or allogenic T cells comprising Tcm cells, e.g., atleast 20%, 30%, 40%, 50% 60%, 70%, 80% of the cells are Tcm cells; atleast 15%, 20%, 25%, 30%, 35% of the Tcm cells are CD4+ and at least15%, 20%, 25%, 30%, 35% of the Tcm cells are CD8+ cells) transduced by avector comprising an expression cassette encoding a chimeric antigenreceptor, wherein chimeric antigen receptor comprises an amino acidsequence selected from SEQ ID NOs: 10, 31-48 and 52. In variousembodiments: the population of human T cells comprise central memory Tcells; the cancer is glioblastoma; and the transduced human T cellswhere prepared by a method comprising obtaining T cells from thepatient, treating the T cells to isolate central memory T cells, andtransducing at least a portion of the central memory cells to with aviral vector comprising an expression cassette encoding a chimericantigen receptor, wherein chimeric antigen receptor comprises an aminoacid sequence selected from SEQ ID NOs: 10, 31-48 and 52.

Also described is: a nucleic acid molecule encoding an polypeptidecomprising an amino acid sequence that is at least 95% identical to anamino acid sequence selected from SEQ ID NO:10 and SEQ ID NOs: 10, 31-48and 52; a nucleic acid molecule encoding an polypeptide comprising anamino acid sequence that is identical to an amino acid sequence selectedfrom SEQ ID NO: 10, 31-48 and 52 except for the presence of no more than5 amino acid substitutions, deletions or insertions; a nucleic acidmolecule encoding an polypeptide comprising an amino acid sequence thatis identical to an amino acid sequence selected from SEQ ID NO:10 andSEQ ID NOs: 10, 31-48 and 52 except for the presence of no more than 5amino acid substitutions; and a nucleic acid molecule encoding anpolypeptide comprising an amino acid sequence that is identical to anamino acid sequence selected from SEQ ID NO:10 and SEQ ID NOs: 10, 31-48and 52 except for the presence of no more than 2 amino acidsubstitutions.

Certain CAR described herein, for example, the IL13(EQ)BBζ CAR and theIL13(EQ)CD28-BBζ CAR, have certain beneficial characteristics comparedto certain other IL13-targeted CAR. For example, they have improvedselectivity for IL13Rα, elicit lower Th2 cytokine production,particularly lower IL13 production.

T cells expressing a CAR targeting IL13Rα2 can be useful in treatment ofcancers such as glioblastoma, as well as other cancer that expressesIL13Rα2 which include but are not limited to medulloblastoma, breastcancer, head and neck cancer, kidney cancer, ovarian cancer and Kaposi'ssarcoma. Thus, this disclosure includes methods for treating cancerusing T cells expressing a CAR described herein.

This disclosure also nucleic acid molecules that encode any of the CARsdescribed herein (e.g., vectors that include a nucleic acid sequenceencoding one of the CARs) and isolated T lymphocytes that express any ofthe CARs described herein.

The CAR described herein can include a spacer region located between theIL13 domain and the transmembrane domain. A variety of different spacerscan be used. Some of them include at least portion of a human Fc region,for example a hinge portion of a human Fc region or a CH3 domain orvariants thereof. Table 1 below provides various spacers that can beused in the CARs described herein.

TABLE 1 Examples of Spacers Name Length Sequence a3   3 aa AAA linker 10 aa GGGSSGGGSG (SEQ ID NO: 14) IgG4 hinge (S→P)  12 aa ESKYGPPCP

CP (SEQ ID NO: 15) (S228P) IgG4 hinge  12 aaESKYGPPCPSCP (SEQ ID NO: 52) IgG4 hinge + linker  22 aaESKYGPPCPPCPGGGSSGGGSG (SEQ ID NO: 16) CD28 hinge  39 aaIEVMYPPPYLDNEKSNGTIIHVKGKHL CPSPLFPGPSKP (SEQ ID NO: 17) CD8 hinge-48 aa 48 aa AKPTTTPAPRPPTPAPTIASQPLSLRPE ACRPAAGGAVHTRGLDFACD (SEQ ID NO: 18)CD8 hinge-45 aa  45 aa TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 19) IgG4(HL-CH3) 129 aaESKYGPPCPPCPGGGSSGGGSGGQPR EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 20) IgG4(L235E, N297Q) 229 aaESKYGPPCPSCPAPEF

GGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVH

AKTKPREEQFN STYRVVSVLTVLHQDWLNGKEYKCK VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLGK (SEQ ID NO: 4)IgG4(S228P, L235E, N297Q) 229 aa ESKYGPPCP

CPAPEF

GGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVH

AKTKPREEQFN STYRVVSVLTVLHQDWLNGKEYKCK VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLGK (SEQ ID NO: 51)IgG4(CH3) 107 aa GQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ EGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 50)Some spacer regions include all or part of an immunoglobulin (e.g.,IgG1, IgG2, IgG3, IgG4) hinge region, i.e., the sequence that fallsbetween the CH1 and CH2 domains of an immunoglobulin, e.g., an IgG4 Fchinge or a CD8 hinge. Some spacer regions include an immunoglobulin CH3domain or both a CH3 domain and a CH2 domain. The immunoglobulin derivedsequences can include one ore more amino acid modifications, forexample, 1, 2, 3, 4 or 5 substitutions, e.g., substitutions that reduceoff-target binding.

An “amino acid modification” refers to an amino acid substitution,insertion, and/or deletion in a protein or peptide sequence. An “aminoacid substitution” or “substitution” refers to replacement of an aminoacid at a particular position in a parent peptide or protein sequencewith another amino acid. A substitution can be made to change an aminoacid in the resulting protein in a non-conservative manner (i.e., bychanging the codon from an amino acid belonging to a grouping of aminoacids having a particular size or characteristic to an amino acidbelonging to another grouping) or in a conservative manner (i.e., bychanging the codon from an amino acid belonging to a grouping of aminoacids having a particular size or characteristic to an amino acidbelonging to the same grouping). Such a conservative change generallyleads to less change in the structure and function of the resultingprotein. The following are examples of various groupings of aminoacids: 1) Amino acids with nonpolar R groups: Alanine, Valine, Leucine,Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine; 2) Aminoacids with uncharged polar R groups: Glycine, Serine, Threonine,Cysteine, Tyrosine, Asparagine, Glutamine; 3) Amino acids with chargedpolar R groups (negatively charged at pH 6.0): Aspartic acid, Glutamicacid; 4) Basic amino acids (positively charged at pH 6.0): Lysine,Arginine, Histidine (at pH 6.0). Another grouping may be those aminoacids with phenyl groups: Phenylalanine, Tryptophan, and Tyrosine.

In certain embodiments, the spacer is derived from an IgG1, IgG2, IgG3,or IgG4 that includes one or more amino acid residues substituted withan amino acid residue different from that present in an unmodifiedspacer. The one or more substituted amino acid residues are selectedfrom, but not limited to one or more amino acid residues at positions220, 226, 228, 229, 230, 233, 234, 235, 234, 237, 238, 239, 243, 247,267, 268, 280, 290, 292, 297, 298, 299, 300, 305, 309, 218, 326, 330,331, 332, 333, 334, 336, 339, or a combination thereof. In thisnumbering scheme, described in greater detail below, the first aminoacid in the IgG4(L235E,N297Q) spacer in Table 1 is 219 and the firstamino acid in the IgG4(HL-CH3) spacer in Table 1 is 219 as is the firstamino acid in the IgG hinge sequence and the IgG4 hinge linker (HL)sequence in Table 1.

In some embodiments, the modified spacer is derived from an IgG1, IgG2,IgG3, or IgG4 that includes, but is not limited to, one or more of thefollowing amino acid residue substitutions: C220S, C226S, S228P, C229S,P230S, E233P, V234A, L234V, L234F, L234A, L235A, L235E, G236A, G237A,P238S, S239D, F243L, P247I, S267E, H268Q, S280H, K290S, K290E, K290N,R292P, N297A, N297Q, S298A, S298G, S298D, S298V, T299A, Y300L, V305I,V309L, E318A, K326A, K326W, K326E, L328F, A330L, A330S, A331S, P331S,1332E, E333A, E333S, E333S, K334A, A339D, A339Q, P396L, or a combinationthereof.

In certain embodiments, the modified spacer is derived from IgG4 regionthat includes one or more amino acid residues substituted with an aminoacid residue different from that present in an unmodified region. Theone or more substituted amino acid residues are selected from, but notlimited to, one or more amino acid residues at positions 220, 226, 228,229, 230, 233, 234, 235, 234, 237, 238, 239, 243, 247, 267, 268, 280,290, 292, 297, 298, 299, 300, 305, 309, 218, 326, 330, 331, 332, 333,334, 336, 339, or a combination thereof.

In some embodiments, the modified spacer is derived from an IgG4 regionthat includes, but is not limited to, one or more of the following aminoacid residue substitutions: 220S, 226S, 228P, 229S, 230S, 233P, 234A,234V, 234F, 234A, 235A, 235E, 236A, 237A, 238S, 239D, 243L, 247I, 267E,268Q, 280H, 290S, 290E, 290N, 292P, 297A, 297Q, 298A, 298G, 298D, 298V,299A, 300L, 305I, 309L, 318A, 326A, 326W, 326E, 328F, 330L, 330S, 331S,331S, 332E, 333A, 333S, 333S, 334A, 339D, 339Q, 396L, or a combinationthereof, wherein the amino acid in the unmodified spacer is substitutedwith the above identified amino acids at the indicated position.

For amino acid positions in immunoglobulin discussed herein, numberingis according to the EU index or EU numbering scheme (Kabat et al. 1991Sequences of Proteins of Immunological Interest, 5th Ed., United StatesPublic Health Service, National Institutes of Health, Bethesda, herebyentirely incorporated by reference). The EU index or EU index as inKabat or EU numbering scheme refers to the numbering of the EU antibody(Edelman et al. 1969 Proc Natl Acad Sci USA 63:78-85).

A variety of transmembrane domains can be used in CAR directed againstIL13Ra2. Table 2 includes examples of suitable transmembrane domains.Where a spacer domain is present, the transmembrane domain is locatedcarboxy terminal to the spacer domain.

TABLE 2 Examples of Transmembrane Domains Name Accession Length SequenceCD3z J04132.1 21 aa LCYLLDGILFIYGVILTALFL (SEQ ID NO: 21) CD28 NM_00613927 aa FWVLVVVGGVLACYSLLVTVAFIIF WV (SEQ ID NO: 22) CD28(M) NM_00613928 aa MFWVLVVVGGVLACYSLLVTVAFII FWV (SEQ ID NO: 22) CD4 M35160 22 aaMALIVLGGVAGLLLFIGLGIFF (SEQ ID NO: 5) CD8tm NM_001768 21 aaIYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 23) CD8tm2 NM_001768 23 aaIYIWAPLAGTCGVLLLSLVITLY (SEQ ID NO: 24) CD8tm3 NM_001768 24 aaIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 25) 41BB NM_001561 27 aaIISFFLALTSTALLFLLFFLTLRF SVV (SEQ ID NO: 26)Many of the CAR described herein include one or more (e.g., two)costimulatory domains. The costimulatory domain(s) are located betweenthe transmembrane domain and the CD3ζ signaling domain. Table 3 includesexamples of suitable costimulatory domains together with the sequence ofthe CD3ζ signaling domain.

TABLE 3 Examples of Costimulatory Domains Name Accession Length SequenceCD3ζ J04132.1 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR CD28 NM_006139 42 aaRSKRSRLLHSDYMNMTPRRPGPTRKH QYPYAPPRDFAAYRS (SEQ ID NO: NO: 27) CD28gg*NM_006139 42 aa RSKRSRGGHSDYMNMTPRRPGPTRKH YQPYAPPRDFAAYRS (SEQ IDNO: 28) 41BB NM_001561 42 aa KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 29) OX40 42 aa ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 30)

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic depiction of IL13(E13Y)-zetakine CAR (Left)composed of the IL13Rα2-specific human IL-13 variant (huIL-13(E13Y)),human IgG4 Fc spacer (huγ₄F_(c)), human CD4 transmembrane (huCD4 tm),and human CD3ζ chain cytoplasmic (huCD3ζ cyt) portions as indicated.Also depicted is a IL13(EQ)BBζ CAR which is the same as theIL13(E13Y)-zetakine with the exception of the two point mutations, L235Eand N297Q indicated in red, that are located in the CH2 domain of theIgG4 spacer, and the addition of a costimulatory 4-1BB cytoplasmicdomain (4-1BB cyt).

FIGS. 2A-C depict certain vectors an open reading frames. A is a diagramof the cDNA open reading frame of the 2670 nucleotideIL13(EQ)BBZ-T2ACD19t construct, where the IL13Rα2-specific ligandIL13(E13Y), IgG4(EQ) Fc hinge, CD4 transmembrane, 4-1BB cytoplasmicsignaling, three-glycine linker, and CD3ζ cytoplasmic signaling domainsof the IL13(EQ)BBZ CAR, as well as the T2A ribosome skip and truncatedCD19 sequences are indicated. The human GM-CSF receptor alpha and CD19signal sequences that drive surface expression of the IL13(EQ)BBζ CARand CD19t are also indicated. B is a diagram of the sequences flanked bylong terminal repeats (indicated by ‘R’) that will integrate into thehost genome. C is a map of the IL13(EQ)BBZ-T2A-CD19t_epHIV7 plasmid.

FIG. 3 depicts the construction of pHIV7.

FIG. 4 depicts the elements of pHIV7.

FIG. 5 depicts a production scheme for IL13(EQ)BBζ/CD19t+ T_(CM).

FIGS. 6A-C depicts the results of flow cytometric analysis of surfacetransgene and T cell marker expression. IL13(EQ)BBζ/CD19t+ T_(CM)HD006.5 and HD187.1 were co-stained with anti-IL13-PE and anti-CD8-FITCto detect CD8+ CAR+ and CD4+ (i.e., CD8 negative) CAR+ cells (A), oranti-CD19-PE and anti-CD4-FITC to detect CD4+ CD19t+ and CD8+ (i.e., CD4negative) CAR+ cells (B). IL13(EQ)BBζ/CD19t+ T_(CM) HD006.5 and HD187.1stained with fluorochromeconjugated anti-CD3, TCR, CD4, CD8, CD62L andCD28 (grey histograms) or isotype controls (black histograms) (C). Inall cases the percentages based on viable lymphocytes (DAPI negative)stained above isotype.

FIGS. 7A-B depict the in vitro functional characterization ofIL13Rα2-specific effector function of IL13(EQ)BBZ+ T_(CM).IL13(EQ)BBZ/CD19t+ T_(CM) HD006.5 and HD187.1 were used as effectors ina 6-hour ⁵¹Cr release assay using a 10:1 E:T ratio based on CD19texpression. The IL13Rα2-positive tumor targets were K562 engineered toexpress IL13Rα2 (K562-IL13Rα2) and primary glioma line PBT030-2, and theIL13Rα2-negative tumor target control was K562 parental line (A).IL13(EQ)BBZ/CD19t+ T_(CM) HD006.5 and HD187.1 were evaluated forantigen-dependent cytokine production following overnight co-culture ata 10:1 E:T ratio with IL13Rα2-positive and negative targets. Cytokinelevels were measured using the Bio-Plex Pro Human Cytokine TH1/TH2 Assaykit and INF-γ are reported (B).

FIGS. 8A-C depict the result of studies demonstrating the regression ofestablished glioma tumor xenografts after adoptive transfer ofIL13(EQ)BBζ/CD19t+ T_(CM). EGFP-ffLuc+ PBT030-2 tumor cells (1×10⁵) werestereotactically implanted into the right forebrain of NSG mice. On day5, mice received either 2×10⁶ IL13(EQ)BBζ/CD19t+ T_(CM) (1.1×10⁶ CAR+;n=6), 2×10⁶ mock TCM (no CAR; n=6) or PBS (n=6). Representative micefrom each group showing relative tumor burden using Xenogen Living Image(A). Quantification of ffLuc flux (photons/sec) shows thatIL13(EQ)BBζ/CD19t+ T_(CM) induce tumor regression as compared tomock-transduced T_(CM) and PBS (#p<0.02, *p<0.001, repeated measuresANOVA) (B). Kaplan Meier survival curve (n=6 per group) demonstratingsignificantly improved survival (p=0.0008; log-rank test) for micetreated with IL13(EQ)BBζ/CD19t+ T_(CM) (C).

FIGS. 9A-C depict the results of studies comparing ant-tumor efficacy ofIL13(EQ)BBZ T_(CM) and IL13-zetakine CTL clones. EGFP-ffLuc+ PBT030-2TSs (1×10⁵) were stereotactically implanted into the right forebrain ofNSG mice. On day 8, mice received either 1.6×10⁶ mock T_(CM) (no CAR),1.0×10⁶ CAR+ IL13(EQ)BBζ T_(CM) (1.6×10⁶ total T cells; 63% CAR),1.0×10⁶ IL13-zetakine CD8+ CTL cl. 2D7 (clonal CAR+), or no treatment(n=6 per group). Representative mice from each group showing relativetumor burden using Xenogen Living Image (A). Linear regression lines ofnatural log of ffLuc flux (photons/sec) over time, P-values are forgroup by time interaction comparisons (B). Kaplan Meier survivalanalysis (n=6 per group) demonstrate significantly improved survival(p=0.02; log-rank test) for mice treated with IL13(EQ)BBζ T_(CM) ascompared to IL13-zetakine CD8+ CTL cl. 2D7 (C).

FIGS. 10A-C depict the results of studies comparing ant-tumor efficacyof IL13(EQ)BBζ T_(CM) and IL13-zetakine CTL clones. EGFP-ffLuc+ PBT030-2TSs (1×10⁵) were stereotactically implanted into the right forebrain ofNSG mice. On day 8, mice received either 1.3×10⁶ mock T_(CM) (no CAR;n=6), 1.0, 0.3 or 0.1×10⁶ CAR+ IL13(EQ)BBζ T_(CM) (78% CAR+; n=6-7),1.0, 0.3 or 0.1×10⁶ IL13-zetakine CD8+ CTL cl. 2D7 (clonal CAR+; n=6-7),or no treatment (n=5). Xenogen imaging of representative mice from eachgroup showing relative tumor burden (A). Linear regression lines ofnatural log of ffLuc flux (photons/sec) shows that IL13(EQ)BBζ T_(CM)achieve superior tumor regression as compared to first-generationIL13-zetakine CTL cl. 2D7, mock T_(CM) and tumor only (B). Average fluxper group at day 27 post tumor injection demonstrating that the 0.1×10⁶IL13(EQ)BBζ T_(CM) dose outperforms the ten-fold higher 1.0×10⁶ dose ofIL13-zetakine CD8+ CTL cl. 2D7 (p=0.043; Welch two sample t-test) (C).

FIG. 11 depicts the results of studies demonstrating IL13(EQ)BBζ Tcmdisplay improved persistence compared IL13-zetakine CTL clones. CD3immunohistochemistry evaluating T cell persistence at the tumor site7-days post T cell infusion. Significant numbers of T cells are detectedfor IL13(EQ)BBζ Tcm (top panel). By contrast, very few viable CD3+IL13-zetakine T cells are detected (bottom panel).

FIGS. 12A-D depict the results of experiments comparing route of CAR+ Tcell delivery (i.c. versus i.v.) for large established tumors.EGFP-ffLuc+ PBT030-2 TSs (1×10⁵) were implanted into the right forebrainof NSG mice. On days 19 and 26, mice were injected i.v. through the tailvein with either 5×10⁶ CAR+ IL13(EQ)BBζ+ Tcm (11.8×10⁶ total cells;n=4), or mock Tcm (11.8×10⁶ cells; n=4). Alternatively, on days 19, 22,26 and 29 mice were injected i.c. with either 1×10⁶ CAR+ IL13(EQ)BBζ+Tcm (2.4×10⁶ total cells; n=4), or mock Tcm (2.4×10⁶ cells; n=5).Average ffLuc flux (photons/sec) over time shows that i.c. deliveredIL13(EQ)BBζ Tcm mediates tumor regression of day 19 tumors. Bycomparison, i.v. delivered T cells do not shown reduction in tumorburden as compared to untreated or mock Tcm controls (A). Kaplan Meiersurvival curve demonstrates improved survival for mice treated i.c.IL13(EQ)BBZ Tcm as compared to mice treated with i.v. administered CAR+Tcm (p=0.0003 log rank test) (B). Representative H&E and CD3 IHC of micetreated i.v. (C) versus i.c. (D) with IL13(EQ)BBZ+ Tcm. CD3+ T cellswere only detected in the i.c. treated group, with no CD3+ cellsdetected in the tumor or surrounding brain parenchyma for i.v. treatedmice.

FIGS. 13A-B depict the results of studies showing that CAR+ T cellinjected intracranially, either intratumoral (i.c.t.) orintraventricular (i.c.v.), can traffic to tumors on the oppositehemisphere. EGFP-ffLuc+ PBT030-2 TSs (1×105) were stereotacticallyimplanted into the right and left forebrains of NSG mice. On day 6, micewere injected i.c. at the right tumor site with 1.0×106 IL13(EQ)BBζ+ Tcm(1.6×106 total cells; 63% CAR; n=4). Schematic of multifocal gliomaexperimental model (A). CD3 IHC showing T cells infiltrating both theright and left tumor sites (B).

FIGS. 14A-C depict the results of a series of studies evaluatingcostimulatory domains of IL13Rα2-specific CAR. Schematic ofIL13Ra2-specific CAR constructs comparing various intracellularendo/signaling domains, including the first generation CD3z CAR lackingcostimulation, versus second generation CARs incorporating either 4-1BBor CD28, versus a third generation CAR containing both CD28 and 41BB.All CAR cassettes also contain the T2A ribosomal skip and truncated CD19(CD19t) sequences as a marker for transduced cells (A). CD4 and CD8 TCMwere lentivirally transduced and CAR-expressing T cells wereimmunomagnetically enriched via anti-CD19. CD19 and IL13 (i.e., CAR)expression levels as measured by flow cytometry (B). Stability of eachCAR construct was determined by dividing the CAR (IL13) meanflourescence intensity (MFI) by that of the transduction marker (CD19t)(C). The 4-1BB containing CARs demonstrated the lowest expression levelsas compared to the CD19t transduction marker.

FIGS. 15A-B depict the results of studies demonstrating thatIL13Rα2-specific CAR containing the 4-1BB costimulatory domain produceless Th1 and Th2 cytokines. The ability of the indicated mock-transducedor CAR-expressing T cells to kill IL13Rα2-expressing PBT030-2 tumor celltargets was determined in a 4-hour 51Cr-release assay at the indicatedeffector:target ratios. Mean % chromium release+S.D. of triplicate wellsare depicted (A). As expected, mock-transduced T cells did notefficiently lyse the targets. In contrast, all CAR-expressing T cellslysed the tumor cells in a similar manner. The indicated mock-transducedor CAR-expressing T cells were co-cultured overnight withIL13Rα2-expressing PBT030-2 tumor cells at a 10:1 ratio and supernatantswere analyzed for IL-13 and IFN-γ levels by cytometric bead array (B).Means+S.D. of triplicate wells are depicted. Interestingly, T cellsexpressing the zeta, 41BB-zeta or CD28-41BB-zeta CARs exhibited lowerantigen-stimulated cytokine production than T cells expressing theCD28-zeta CAR.

FIGS. 16A-C depict the results of a series of studies of the in vivoefficacy of IL13Rα2-specific CARs. NSG mice received an intracranialinjection of ffLuc+ PBT030-2 tumor cells on day 0, and were randomizedinto 6 groups (n=9-10 mice per group) for i.c. treatment with either PBS(Tumor Only), mock-transduced T cells or T cells expressing theindicated IL13Rα2-specific CAR on day 8. Quantitative bioluminescenceimaging was then carried out to monitor tumor growth over time.Bioluminescence images for representative mice in each group (A).Mean+S.E. of total flux levels of luciferase activity over time in eachgroup (B). Flux levels for each mouse at Day 27. All groups treated withIL13Rα2-specific CAR T cells, except those treated with T cellsexpressing the CD28-CAR, show statistically-significant reduction intumor volume compared to mice treated with mock-transduced T cells (C)

FIGS. 17A-B depict the amino acid sequence of IL13(EQ)BBζ/CD19t+ (SEQ IDNO:10).

FIGS. 18A-O depict a sequence comparison of IL13(EQ)41BBζ[IL13{EQ}41BBζT2A-CD19t_epHIV7; pF02630] (SEQ ID NO:12) and CD19Rop_epHIV7 (pJ01683)(SEQ ID NO:13).

FIG. 19 depicts the amino acid sequence of IL13(EmY)-CD8h3-CD8tm2-41BBZeta (SEQ ID NO:31 with GMSCFRa signal peptide; SEQ ID NO:39 withoutGMSCFRa signal peptide).

FIG. 20 depicts the amino acid sequence ofIL13(EmY)-CD8h3-CD28tm-CD28gg-41BB-Zeta (SEQ ID NO:32 with GMSCFRasignal peptide; SEQ ID NO:40 without GMSCFRa signal peptide).

FIG. 21 depicts the amino acid sequence ofIL13(EmY)-IgG4(HL-CH3)-CD4tm-41BB-Zeta (SEQ ID NO:33 with GMSCFRa signalpeptide; SEQ ID NO:41 without GMSCFRa signal peptide).

FIG. 22 depicts the amino acid sequence ofIL13(EmY)-IgG4(L235E,N297Q)-CD8tm-41BB-Zeta (SEQ ID NO:34 with GMSCFRasignal peptide; SEQ ID NO:42 without GMSCFRa signal peptide).

FIG. 23 depicts the amino acid sequence ofIL13(EmY)-Linker-CD28tm-CD28gg-41BB-Zeta (SEQ ID NO:35 with GMSCFRasignal peptide; SEQ ID NO:43without GMSCFRa signal peptide).

FIG. 24 depicts the amino acid sequence ofIL13(EmY)-HL-CD28m-CD28gg-41BB-Zeta (SEQ ID NO:36 with GMSCFRa signalpeptide; SEQ ID NO:44 without GMSCFRa signal peptide).

FIG. 25 depicts the amino acid sequence ofIL13(EmY)-IgG4(HL-CH3)-CD28tm-CD28gg-41BB-Zeta (SEQ ID NO:37 withGMSCFRa signal peptide; SEQ ID NO:45 without GMSCFRa signal peptide).

FIG. 26 depicts the amino acid sequence of IL13(EmY)IgG4(L235E,N297Q)-CD28tm-CD28gg-41BB-Zeta (SEQ ID NO:38 with GMSCFRasignal peptide; SEQ ID NO:46 without GMSCFRa signal peptide).

FIG. 27 depicts the amino acid sequence of IL13(EmY)-CD8h3-CD8tm-41BBZeta (SEQ ID NO:47 with GMSCFRa signal peptide; SEQ ID NO:48 withoutGMSCFRa signal peptide).

DETAILED DESCRIPTION

Described below is the structure, construction and characterization ofvarious IL13Rα2-specific chimeric antigen receptors. A chimeric antigen(CAR) is a recombinant biomolecule that contains, at a minimum, anextracellular recognition domain, a transmembrane region, and anintracellular signaling domain. The term “antigen,” therefore, is notlimited to molecules that bind antibodies, but to any molecule that canbind specifically to a target. For example, a CAR can include a ligandthat specifically binds a cell surface receptor. The extracellularrecognition domain (also referred to as the extracellular domain orsimply by the recognition element which it contains) comprises arecognition element that specifically binds to a molecule present on thecell surface of a target cell. The transmembrane region anchors the CARin the membrane. The intracellular signaling domain comprises thesignaling domain from the zeta chain of the human CD3 complex andoptionally comprises one or more costimulatory signaling domains. CARscan both to bind antigen and transduce T cell activation, independent ofMEW restriction. Thus, CARs are “universal” immunoreceptors which cantreat a population of patients with antigen-positive tumors irrespectiveof their HLA genotype. Adoptive immunotherapy using T lymphocytes thatexpress a tumor-specific CAR can be a powerful therapeutic strategy forthe treatment of cancer.

One IL13Rα2-specific CAR described herein is referred to as IL13(EQ)BBζ.This CAR includes a variety of important features including: a IL13α2ligand having an amino acid change that improves specificity of bidingto IL13α2; the domain of CD137 (4-1BB) in series with CD3ζ to providebeneficial costimulation; and an IgG4 Fc region that is mutated at twosites within the CH2 region (L235E; N297Q) in a manner that reducesbinding by Fc receptors (FcRs). Other CAR described herein contain asecond costimulatory domain.

In some cases the CAR described herein, including the IL13(EQ)BBζ CARcan be produced using a vector in which the CAR open reading frame isfollowed by a T2A ribosome skip sequence and a truncated CD19 (CD19t),which lacks the cytoplasmic signaling tail (truncated at amino acid323). In this arrangement, co-expression of CD19t provides an inert,non-immunogenic surface marker that allows for accurate measurement ofgene modified cells, and enables positive selection of gene-modifiedcells, as well as efficient cell tracking and/or imaging of thetherapeutic T cells in vivo following adoptive transfer. Co-expressionof CD19t provides a marker for immunological targeting of the transducedcells in vivo using clinically available antibodies and/or immunotoxinreagents to selectively delete the therapeutic cells, and therebyfunctioning as a suicide switch.

Gliomas, express IL13 receptors, and in particular, high-affinity IL13receptors. However, unlike the IL13 receptor, glioma cells overexpress aunique IL13Rα2 chain capable of binding IL13 independently of therequirement for IL4Rβ or γc44. Like its homolog IL4, IL13 has pleotropicimmunoregulatory activity outside the CNS. Both IL13 and IL4 stimulateIgE production by B lymphocytes and suppress pro-inflammatory cytokineproduction by macrophages.

Detailed studies using autoradiography with radiolabeled IL13 havedemonstrated abundant IL13 binding on nearly all malignant gliomatissues studied. This binding is highly homogeneous within tumorsections and in single cell analysis. However, molecular probe analysisspecific for IL13Rα2 mRNA did not detect expression of theglioma-specific receptor by normal brain elements and autoradiographywith radiolabeled IL13 also could not detect specific IL13 binding inthe normal CNS. These studies suggest that the shared IL13Rα1/IL4β/γcreceptor is not expressed detectably in the normal CNS. Therefore,IL13Rα2 is a very specific cell-surface target for glioma and is asuitable target for a CAR designed for treatment of a glioma.

Binding of IL13-based therapeutic molecules to the broadly expressedIL13Rα1/IL4β/γc receptor complex, however, has the potential ofmediating undesired toxicities to normal tissues outside the CNS, andthus limits the systemic administration of these agents. An amino acidsubstitution in the IL13 alpha helix A at amino acid 13 of tyrosine forthe native glutamic acid selectively reduces the affinity of IL13 to theIL13Rα1/IL4β/γc receptor. Binding of this mutant (termed IL13(E13Y)) toIL13Rα2, however, was increased relative to wild-type IL13. Thus, thisminimally altered IL13 analog simultaneously increases IL13'sspecificity and affinity for glioma cells. Therefore, CAR describedherein include an IL13 containing a mutation (E to Y or E to some otheramino acid such as K or R or L or V) at amino acid 13 (according to thenumbering of Debinski et al. 1999 Clin Cancer Res 5:3143s). IL13 havingthe natural sequence also may be used, however, and can be useful,particularly in situations where the modified T cells are to be locallyadministered, such as by injection directly into a tumor mass.

The CAR described herein can be produced by any means known in the art,though preferably it is produced using recombinant DNA techniques.Nucleic acids encoding the several regions of the chimeric receptor canbe prepared and assembled into a complete coding sequence by standardtechniques of molecular cloning known in the art (genomic libraryscreening, PCR, primer-assisted ligation, site-directed mutagenesis,etc.) as is convenient. The resulting coding region is preferablyinserted into an expression vector and used to transform a suitableexpression host cell line, preferably a T lymphocyte cell line, and mostpreferably an autologous T lymphocyte cell line.

Various T cell subsets isolated from the patient, including unselectedPBMC or enriched CD3 T cells or enriched CD3 or memory T cell subsets,can be transduced with a vector for CAR expression. Central memory Tcells are one useful T cell subset. Central memory T cell can beisolated from peripheral blood mononuclear cells (PBMC) by selecting forCD45RO+/CD62L+ cells, using, for example, the CliniMACS® device toimmunomagnetically select cells expressing the desired receptors. Thecells enriched for central memory T cells can be activated withanti-CD3/CD28, transduced with, for example, a SIN lentiviral vectorthat directs the expression of an IL13Rα2-specific CAR (e.g.,IL13(EQ)BBζ) as well as a truncated human CD19 (CD19t), anon-immunogenic surface marker for both in vivo detection and potentialex vivo selection. The activated/genetically modified central memory Tcells can be expanded in vitro with IL-2/IL-15 and then cryopreserved.

EXAMPLE 1 Construction and Structure of an IL13Rα2-Specific CAR

The structure of a useful IL13Rα2-specific CAR is described below. Thecodon optimized CAR sequence contains a membrane-tethered IL-13 ligandmutated at a single site (E13Y) to reduce potential binding to IL13Rα1,an IgG4 Fc spacer containing two mutations (L235E; N297Q) that greatlyreduce Fc receptor-mediated recognition models, a CD4 transmembranedomain, a costimulatory 4-1BB cytoplasmic signaling domain, and a CD3ζcytoplasmic signaling domain. A T2A ribosome skip sequence separatesthis IL13(EQ)BBζ CAR sequence from CD19t, an inert, non-immunogenic cellsurface detection/selection marker. This T2A linkage results in thecoordinate expression of both IL13(EQ)BBζ and CD19t from a singletranscript. FIG. 1A is a schematic drawing of the 2670 nucleotide openreading frame encoding the IL13(EQ)BBZ-T2ACD19t construct. In thisdrawing, the IL13Rα2-specific ligand IL13(E13Y), IgG4(EQ) Fc, CD4transmembrane, 4-1BB cytoplasmic signaling, three-glycine linker, andCD3ζ cytoplasmic signaling domains of the IL13(EQ)BBZ CAR, as well asthe T2A ribosome skip and truncated CD19 sequences are all indicated.The human GM-CSF receptor alpha and CD19 signal sequences that drivesurface expression of the IL13(EQ)BBZ CAR and CD19t are also indicated.Thus, the IL13(EQ)BBZ-T2ACD19t construct includes a IL13Rα2-specific,hinge-optimized, costimulatory chimeric immunoreceptor sequence(designated IL13(EQ)BBZ), a ribosome-skip T2A sequence, and a CD19tsequence.

The IL13(EQ)BBZ sequence was generated by fusion of the human GM-CSFreceptor alpha leader peptide with IL13(E13Y) ligand 5L235E/N297Q-modified IgG4 Fc hinge (where the double mutation interfereswith FcR recognition), CD4 transmembrane, 4-1BB cytoplasmic signalingdomain, and CD3ζ cytoplasmic signaling domain sequences. This sequencewas synthesized de novo after codon optimization. The T2A sequence wasobtained from digestion of a T2A-containing plasmid. The CD19t sequencewas obtained from that spanning the leader peptide sequence to thetransmembrane components (i.e., basepairs 1-972) of a CD19-containingplasmid. All three fragments, 1) IL13(EQ)BBZ, 2) T2A, and 3) CD19t, werecloned into the multiple cloning site of the epHIV7 lentiviral vector.When transfected into appropriate cells, the vector integrates thesequence depicted schematically in FIG. 1B into the host cells genome.FIG. 1C provides a schematic drawing of the 9515 basepairIL13(EQ)BBZ-T2A-CD19t_epHIV7 plasmid itself.

As shown schematically in FIG. 2, IL13(EQ)BBZ CAR differs in severalimportant respects from a previously described IL13Rα2-specific CARreferred to as IL13(E13Y)-zetakine (Brown et al. 2012 Clinical CancerResearch 18:2199). The IL13(E13Y)-zetakine is composed of theIL13Rα2-specific human IL-13 mutein (huIL-13(E13Y)), human IgG4 Fcspacer (huγ4Fc), human CD4 transmembrane (huCD4 tm), and human CD3ζchain cytoplasmic (huCD3ζ cyt) portions as indicated. In contrast, theIL13(EQ)BBζ) has two point mutations, L235E and N297Q that are locatedin the CH2 domain of the IgG4 spacer, and a costimulatory 4-1BBcytoplasmic domain (4-1BB cyt).

EXAMPLE 2 Construction and Structure of epHIV7 used for Expression of anIL13Rα2-Specific CAR

The pHIV7 plasmid is the parent plasmid from which the clinical vectorIL13(EQ)BBZ-T2A-CD19t_epHIV7 was derived in the T cell TherapeuticsResearch Laboratory (TCTRL) at City of Hope (COH). The epHIV7 vectorused for expression of the CAR was produced from pHIV7 vector.Importantly, this vector uses the human EF1 promoter to drive expressionof the CAR. Both the 5′ and 3′ sequences of the vector were derived frompv653RSN as previously derived from the HXBc2 provirus. The polypurinetract DNA flap sequences (cPPT) were derived from HIV-1 strain pNL4-3from the NIH AIDS Reagent Repository. The woodchuck post-transcriptionalregulatory element (WPRE) sequence was previously described.

Construction of pHIV7 is schematically depicted in FIG. 3. Briefly,pv653RSN, containing 653 bp from gag-pol plus 5′ and 3′ long-terminalrepeats (LTRs) with an intervening SL3-neomycin phosphotransferase gene(Neo), was subcloned into pBluescript, as follows: In Step 1, thesequences from 5′ LTR to rev-responsive element (RRE) made p5′HIV-1 51,and then the 5′ LTR was modified by removing sequences upstream of theTATA box, and ligated first to a CMV enhancer and then to the SV40origin of replication (p5′HIV-2). In Step 2, after cloning the 3′ LTRinto pBluescript to make p3′HIV-1, a 400-bp deletion in the 3′ LTRenhancer/promoter was made to remove cis-regulatory elements in HIV U3and form p3′HIV-2. In Step 3, fragments isolated from the p5′HIV-3 andp3′HIV-2 were ligated to make pHIV-3. In Step 4, the p3′HIV-2 wasfurther modified by removing extra upstream HIV sequences to generatep3′HIV-3 and a 600-bp BamHI-SalI fragment containing WPRE was added top3′HIV-3 to make the p3′HIV-4. In Step 5, the pHIV-3 RRE was reduced insize by PCR and ligated to a 5′ fragment from pHIV-3 (not shown) and tothe p3′HIV-4, to make pHIV-6. In Step 6, a 190-bp BglII-BamHI fragmentcontaining the cPPT DNA flap sequence from HIV-1 pNL4-3 (55) wasamplified from pNL4-3 and placed between the RRE and the WPRE sequencesin pHIV6 to make pHIV-7. This parent plasmid pHIV7-GFP (GFP, greenfluorescent protein) was used to package the parent vector using afour-plasmid system.

A packaging signal, psi ψ, is required for efficient packaging of viralgenome into the vector. The RRE and WPRE enhance the RNA transcripttransport and expression of the transgene. The flap sequence, incombination with WPRE, has been demonstrated to enhance the transductionefficiency of lentiviral vector in mammalian cells.

The helper functions, required for production of the viral vector), aredivided into three separate plasmids to reduce the probability ofgeneration of replication competent lentivirus via recombination: 1)pCgp encodes the gag/pol protein required for viral vector assembly; 2)pCMV-Rev2 encodes the Rev protein, which acts on the RRE sequence toassist in the transportation of the viral genome for efficientpackaging; and 3) pCMV-G encodes the glycoprotein of thevesiculo-stomatitis virus (VSV), which is required for infectivity ofthe viral vector.

There is minimal DNA sequence homology between the pHIV7 encoded vectorgenome and the helper plasmids. The regions of homology include apackaging signal region of approximately 600 nucleotides, located in thegag/pol sequence of the pCgp helper plasmid; a CMV promoter sequence inall three helper plasmids; and a RRE sequence in the helper plasmidpCgp. It is highly improbable that replication competent recombinantvirus could be generated due to the homology in these regions, as itwould require multiple recombination events. Additionally, any resultingrecombinants would be missing the functional LTR and tat sequencesrequired for lentiviral replication.

The CMV promoter was replaced by the EF1α-HTLV promoter (EF1p), and thenew plasmid was named epHIV7 (FIG. 4). The EF1p has 563 bp and wasintroduced into epHIV7 using NruI and NheI, after the CMV promoter wasexcised.

The lentiviral genome, excluding gag/pol and rev that are necessary forthe pathogenicity of the wild-type virus and are required for productiveinfection of target cells, has been removed from this system. Inaddition, the IL13(EQ)BBZ-T2ACD19t_epHIV7 vector construct does notcontain an intact 3′LTR promoter, so the resulting expressed and reversetranscribed DNA proviral genome in targeted cells will have inactiveLTRs. As a result of this design, no HIV-I derived sequences will betranscribed from the provirus and only the therapeutic sequences will beexpressed from their respective promoters. The removal of the LTRpromoter activity in the SIN vector is expected to significantly reducethe possibility of unintentional activation of host genes (56). Table 4summarizes the various regulator elements present inIL13(EQ)BBZ-T2ACD19t_epHIV7.

TABLE 4 Functional elements of IL13(EQ)41BBZ-T2A-CD19t_epHIV7 RegulatoryLocation Elements (Nucleotide and Genes Numbers) Comments U5  87-171 5′Unique sequence psi 233-345 Packaging signal RRE  957-1289Rev-responsive element flap 1290-1466 Contains polypurine track sequenceand central termination sequence to facilitate nuclear import ofpre-integration complex EF1p Promoter 1524-2067 EF1-alpha EukaryoticPromoter sequence driving expression of CD19Rop IL13-IgG4 (EQ)-2084-4753 Therapeutic insert 41BB-Zeta-T2A- CD19t WPRE 4790-5390Woodchuck hepatitis virus derived regulatory element to enhance viralRNA transportation delU3 5405-5509 3′ U3 with deletion to generate SINvector R 5510-5590 Repeat sequence within LTR U5 5591-5704 3′ U5sequence in LTR Amp^(R) 6540-7398 Ampicillin-resistance gene CoE1 ori7461-8342 Replication origin of plasmid SV40 ori 8639-8838 Replicationorigin of SV40 CMV promoter 8852-9451 CMV promoter to generate viralgenome RNA R 9507-86  Repeat sequence within LTR

EXAMPLE 3 Production of Vectors for Transduction of Patient T Cells

For each plasmid (IL13(EQ)BBZ-T2A-CD19t_epHIV7; pCgp; pCMV-G; andpCMV-Rev2), a seed bank is generated, which is used to inoculate thefermenter to produce sufficient quantities of plasmid DNA. The plasmidDNA is tested for identity, sterility and endotoxin prior to its use inproducing lentiviral vector.

Briefly, cells were expanded from the 293T working cell (WCB), which hasbeen tested to confirm sterility and the absence of viral contamination.A vial of 293T cells from the 293T WCB was thawed. Cells were grown andexpanded until sufficient numbers of cells existed to plate anappropriate number of 10 layer cell factories (CFs) for vectorproduction and cell train maintenance. A single train of cells can beused for production.

The lentiviral vector was produced in sub-batches of up to 10 CFs. Twosub-batches can be produced in the same week leading to the productionof approximately 20 L of lentiviral supernatant/week. The materialproduced from all sub-batches were pooled during the downstreamprocessing phase, in order to produce one lot of product. 293T cellswere plated in CFs in 293T medium (DMEM with 10% FBS). Factories wereplaced in a 37° C. incubator and horizontally leveled in order to get aneven distribution of the cells on all the layers of the CF. Two dayslater, cells were transfected with the four lentiviral plasmidsdescribed above using the CaPO4 method, which involves a mixture ofTris:EDTA, 2M CaCl2, 2× HBS, and the four DNA plasmids. Day 3 aftertransfection, the supernatant containing secreted lentiviral vectors wascollected, purified and concentrated. After the supernatant was removedfrom the CFs, End-of-Production Cells were collected from each CF. Cellswere trypsinized from each factory and collected by centrifugation.Cells were resuspended in freezing medium and cryopreserved. These cellswere later used for replication-competent lentivirus (RCL) testing.

To purify and formulate vectors crude supernatant was clarified bymembrane filtration to remove the cell debris. The host cell DNA andresidual plasmid DNA were degraded by endonuclease digestion(Benzonase®). The viral supernatant was clarified of cellular debrisusing a 0.45 μm filter. The clarified supernatant was collected into apre-weighed container into which the Benzonase® is added (finalconcentration 50 U/mL). The endonuclease digestion for residual plasmidDNA and host genomic DNA as performed at 37° C. for 6 h. The initialtangential flow ultrafiltration (TFF) concentration of theendonuclease-treated supernatant was used to remove residual lowmolecular weight components from the crude supernatant, whileconcentrating the virus ˜20 fold. The clarified endonuclease-treatedviral supernatant was circulated through a hollow fiber cartridge with aNMWCO of 500 kD at a flow rate designed to maintain the shear rate at˜4,000 sec-1 or less, while maximizing the flux rate. Diafiltration ofthe nuclease-treated supernatant was initiated during the concentrationprocess to sustain the cartridge performance. An 80% permeatereplacement rate was established, using 4% lactose in PBS as thediafiltration buffer. The viral supernatant was brought to the targetvolume, representing a 20-fold concentration of the crude supernatant,and the diafiltration was continued for 4 additional exchange volumes,with the permeate replacement rate at 100%.

Further concentration of the viral product was accomplished by using ahigh speed centrifugation technique. Each sub-batch of the lentiviruswas pelleted using a Sorvall RC-26 plus centrifuge at 6000 RPM (6,088RCF) at 6° C. for 16-20 h. The viral pellet from each sub-batch was thenreconstituted in a 50 mL volume with 4% lactose in PBS. Thereconstituted pellet in this buffer represents the final formulation forthe virus preparation. The entire vector concentration process resultedin a 200-fold volume reduction, approximately. Following the completionof all of the sub-batches, the material was then placed at −80° C.,while samples from each sub-batch were tested for sterility. Followingconfirmation of sample sterility, the sub-batches were rapidly thawed at37° C. with frequent agitation. The material was then pooled andmanually aliquoted in the Class II Type A/B3 biosafety cabinet in theviral vector suite. A fill configuration of 1 mL of the concentratedlentivirus in sterile USP class 6, externally threaded O-ring cryovialswas used. Center for Applied Technology Development (CATD)'s QualitySystems (QS) at COH released all materials according to the Policies andStandard Operating Procedures for the CBG and in compliance with currentGood Manufacturing Practices (cGMPs).

To ensure the purity of the lentiviral vector preparation, it was testedfor residual host DNA contaminants, and the transfer of residual hostand plasmid DNA. Among other tests, vector identity was evaluated byRT-PCR to ensure that the correct vector is present. All releasecriteria were met for the vector intended for use in this study.

EXAMPLE 4 Preparation of T cells Suitable for Use in ACT

T lymphocytes are obtained from a patient by leukopheresis, and theappropriate allogenic or autologous T cell subset, for example, CentralMemory T cells (T_(CM)), are genetically altered to express the CAR,then administered back to the patient by any clinically acceptablemeans, to achieve anti-cancer therapy.

An outline of the manufacturing strategy for T_(CM) is depicted in FIG.8 (Manufacturing schema for IL13(EQ)BBζ/CD19t+ T_(CM)). Specifically,apheresis products obtained from consented research participants areficolled, washed and incubated overnight. Cells are then depleted ofmonocyte, regulatory T cell and naïve T cell populations using GMP gradeanti-CD14, anti-CD25 and anti-CD45RA reagents (Miltenyi Biotec) and theCliniMACS™ separation device. Following depletion, negative fractioncells are enriched for CD62L+ T_(CM) cells using DREG56-biotin (COHclinical grade) and anti-biotin microbeads (Miltenyi Biotec) on theCliniMACS™ separation device.

Following enrichment, T_(CM) cells are formulated in complete X-Vivo15plus 50 IU/mL IL-2 and 0.5 ng/mL IL-15 and transferred to a Teflon cellculture bag, where they are stimulated with Dynal ClinEx™ Vivo CD3/CD28beads. Up to five days after stimulation, cells are transduced withIL13(EQ)BBZ-T2A-CD19t_epHIV7 lentiviral vector at a multiplicity ofinfection (MOI) of 1.0 to 0.3. Cultures are maintained for up to 42 dayswith addition of complete X-Vivo15 and IL-2 and IL-15 cytokine asrequired for cell expansion (keeping cell density between 3×10⁵ and2×10⁶ viable cells/mL, and cytokine supplementation every Monday,Wednesday and Friday of culture). Cells typically expand toapproximately 10⁹ cells under these conditions within 21 days. At theend of the culture period cells are harvested, washed twice andformulated in clinical grade cryopreservation medium (Cryostore CS5,BioLife Solutions).

On the day(s) of T cell infusion, the cryopreserved and released productis thawed, washed and formulated for re-infusion. The cryopreservedvials containing the released cell product are removed from liquidnitrogen storage, thawed, cooled and washed with a PBS/2% human serumalbumin (HSA) Wash Buffer. After centrifugation, the supernatant isremoved and the cells resuspended in a Preservative-Free Normal Saline(PFNS)/2% HSA infusion diluent. Samples are removed for quality controltesting.

Two qualification runs on cells procured from healthy donors wereperformed using the manufacturing platform described above. Eachpreclinical qualification run product was assigned a human donor (HD)number—HD006.5 and HD187.1. Importantly, as shown in Table 5, thesequalification runs expanded >80 fold within 28 days and the expandedcells expressed the IL13(EQ)BBγ/CD19t transgenes.

TABLE 5 Summary of Expression Data from Pre- clinical Qualification RunProduct Cell Product CAR CD19 CD4+ CD8+ Fold Expansion HD006.5 20% 22%24% 76%  84-fold (28 days) Hd187.1 18% 25% 37% 63% 259-fold (28 days)

EXAMPLE 5 Flow Cytometric Analysis of Surface Transgene and T CellMarker Expression in IL13(EQ)BBγ/CD19t+ T_(CM)

The two preclinical qualification run products described in Example 4were used in pre-clinical studies to as described below. FIGS. 6A-Cdepict the results of flow cytometric analysis of surface transgene andT cell marker expression. IL13(EQ)BBγ/CD19t+ T_(CM) HD006.5 and HD187.1were co-stained with anti-IL13-PE and anti-CD8-FITC to detect CD8+ CAR+and CD4+ (i.e., CD8 negative) CAR+ cells (FIG. 6A), or anti-CD19-PE andanti-CD4-FITC to detect CD4+ CD19t+ and CD8+ (i.e., CD4 negative) CAR+cells (FIG. 6B). IL13(EQ)BBγ/CD19t+ T_(CM) HD006.5 and HD187.1 werestained with fluorochrome-conjugated anti-CD3, TCR, CD4, CD8, CD62L andCD28 (grey histograms) or isotype controls (black histograms). (FIG.6C). In each of FIGS. 6A-C, the percentages indicated are based onviable lymphocytes (DAPI negative) stained above isotype.

EXAMPLE 6 Effector Activity of IL13(EQ)BBγ/CD19t+ T_(CM)

The effector activity of IL13(EQ)BBζ/CD19t+ T_(CM) was assessed and theresults of this analysis are depicted in FIGS. 7A-B. Briefly,IL13(EQ)BBγ/CD19t+ T_(CM) HD006.5 and HD187.1 were used as effectors ina 6-hour 51Cr-release assay using a 10E:1T ratio based on CD19texpression. The IL13Rα2-positive tumor targets were K562 engineered toexpress IL13Rα2 (K562-IL13Rα2) and primary glioma line PBT030-2, and theIL13Rα2-negative tumor target control was the K562 parental line (FIG.7A). IL13(EQ)BBγ/CD19t+ HD006.5 and HD187.1 were evaluated forantigen-dependent cytokine production following overnight co-culture ata 10E:1T ratio with the same IL13Ra2-positive and negative targets asdescribed in above. Cytokine levels were measured using the Bio-Plex ProHuman Cytokine TH1/TH2 Assay kit and INF-γ levels are depicted (FIG.7B).

EXAMPLE 7 In Vivo Anti-Tumor Activity of IL13(EQ)BBγ/CD19t+ T_(CM)

The studies described below demonstrate that IL13(EQ)BBγ/CD19t+ T_(CM)exhibit anti-tumor efficacy in in vivo mouse models. Specifically, wehave evaluated the anti-tumor potency of IL13(EQ)BBγ/CD19t+ T_(CM)against the IL13Rα2+ primary low-passage glioblastoma tumor sphere linePBT030-2, which has been engineered to express both EGFP and fireflyluciferase (ffLuc) reporter genes (PBT030-2 EGFP:ffLuc) (6). A panel ofprimary lines (PBT) from patient glioblastoma specimens grown as tumorspheres (TSs) in serum-free media. These expanded TS lines exhibit stemcell-like characteristics, including expression of stem cell markers,multilineage differentiation and capacity to initiate orthotopic tumorsin immunocompromised mice (NSG) at low cell numbers. The PBT030-2EGFP:ffLuc TS-initiated xenograft model (0.1×10⁶ cells; 5 dayengraftment) has been previously used to evaluate in vivo anti-tumoractivity in NSG mice of IL13Rα2-specific CAR expressing T cells, wherebythree injections of 2×10⁶ cytolytic T lymphocytes (CTLs) over a courseof 2 weeks were shown to reduce tumor growth. However, in thoseexperiments the majority of the PBT030-2 tumors eventually recurred. Bycomparison, a single injection of IL13(EQ)BBγ/CD19t+ T_(CM) (1.1×10⁶CAR+ T_(CM); 2×10⁶ total TCM) exhibited robust anti-tumor activityagainst PBT030-2 EGFP:ffLuc TS-initiated tumors (0.1×10⁶ cells; 5 dayengraftment) as shown in FIGS. 8A-C. As compared to NSG mice treatedwith either PBS or mock transduced T_(CM) (no CAR), IL13(EQ)BBγ/CD19t+T_(CM) significantly reduce ffLuc flux (p<0.001 at >18-days) andsignificantly improve survival (p=0.0008).

Briefly, EGFP-ffLuc+ PBT030-2 tumor cells (1×10⁵) were stereotacticallyimplanted into the right forebrain of NSG mice. On day 5, mice receivedeither 2×10⁶ IL13(EQ)BBγ/CD19t+ T_(CM) (1.1×106 CAR+; n=6), 2×10⁶ mockT_(CM) (no CAR; n=6) or PBS (n=6). FIG. 8A depicts representative micefrom each group showing relative tumor burden using Xenogen LivingImage. Quantification of ffLuc flux (photons/sec) shows thatIL13(EQ)BBζ/CD19t+ T_(CM) induce tumor regression as compared tomock-transduced T_(CM) and PBS (#p<0.02, *p<0.001, repeated measuresANOVA) (FIG. 8B). As shown in FIG. 8C, a Kaplan Meier survival curve(n=6 per group) demonstrates significantly improved survival (p=0.0008;log-rank test) for mice treated with IL13(EQ)BBγ/CD19t+ T_(CM).

EXAMPLE 8 Comparison of IL13(EQ)BBζ+ Tcm and Non-Tcm IL13-zetakine CD8+CTL Clones in Antitumor Efficacy and T Cell Persistence

The studies described below compare IL13(EQ)BBζ+ Tcm and a previouslycreated IL13Rα2-specific human CD8+ CTLs (IL13-zetakine CD8+ CTL(described in Brown et al. 2012 Clin Cancer Res 18:2199 and Kahlon etal. 2004 Cancer Res 64:9160). The IL13-zetakine uses a CD3ζ stimulatorydomain, lacks a co-stimulatory domain and uses the same IL13 variant asIL13(EQ)BBζ+.

A panel of primary lines (PBT) from patient glioblastoma specimens grownas tumor spheres (TSs) in serum-free media was generated (Brown et al.2012 Clin Cancer Res 18:2199; Brown et al. 2009 Cancer Res 69:8886).These expanded TS lines exhibit stem cell-like characteristics,including expression of stem cell markers, multi-lineage differentiationand capacity to initiate orthotopic tumors in immunocompromised mice(NSG) at low cell numbers. The IL13Rα2+ primary low-passage glioblastomaTS line PBT030-2, which has been engineered to express both EGFP andfirefly luciferase (ffLuc) reporter genes (PBT030-2 EGFP:ffLuc) (Brownet al. 2012 Clin Cancer Res 18:2199) was used for the experimentsoutlined below.

First, a single dose (1×10⁶ CAR T cells) of IL13(EQ)BBζ+ Tcm product wascompared to IL13-zetakine CD8+ CTL clones evaluated against day 8PBT030-2 EGFP:ffuc TS-initiated xenografts (0.1×10⁶ cells). While bothIL13Rα2-specific CAR T cells (IL13-zetakine CTL and IL13(EQ)BBζ Tcm)demonstrated antitumor activity against established PBT030-2 tumors ascompared to untreated and mock Tcm (CAR-negative) controls (FIGS. 9A and9B), IL13(EQ)BBZ+ Tcm mediated significantly improved survival anddurable tumor remission with mice living >150 days as compared to ourfirst-generation IL13-zetakine CD8+ CTL clones (FIG. 9C).

To further compare the therapeutic effectiveness of these twoIL13Rα2-CAR T cell products, a dose titration of 1.0, 0.3 and 0.1×10⁶CAR T cells against day 8 PBT030-2 EGFP:ffuc TS-initiated tumors wasperformed (FIGS. 10A-C). The highest dose (1×10⁶) of IL13-zetakine CD8+CTL cl. 2D7 mediated antitumor responses as measured by Xenogen flux in3 of 6 animals (FIG. 10C), but no significant antitumor responses wereobserved at lower CAR T cell doses. By comparison, injection ofIL13(EQ)BBζ+ Tcm product mediated complete tumor regression in themajority of mice at all dose levels, including treatment with as few as0.1×10⁶ CAR T cells. These data demonstrate that IL13(EQ)BBζ+ Tcm is atleast 10-fold more potent than IL13-zetakine CD8+ CTL clones inantitumor efficacy. The improved anti-tumor efficacy of is due toimproved T cell persistence in the tumor microenvironment. Evaluation ofCD3+ T cells 7-days post i.c. injection revealed significant numbers ofIL13(EQ)BBζ+ Tcm in the tumor microenvironment, whereas very fewfirst-generation IL13-zeta CTLs were present (FIG. 11).

EXAMPLE 9 Comparison of CAR T Cell Delivery Route for Treatment of LargeTS-Initiated PBT Tumors

Described below are studies that compare the route of delivery,intraveneous (i.v.) or intracranial (i.c.), on antitumor activityagainst invasive primary PBT lines. In pilot studies (data not shown),it was unexpectedly observed that i.v. administered IL13(EQ)BBζ+ Tcmprovided no therapeutic benefit as compared to PBS for the treatment ofsmall (day 5) PBT030-2 EGFP:ffLuc tumors. This is in contrast to therobust therapeutic efficacy observed with i.c. administered CAR+ Tcells. Reasoning that day 5 PBT030-2 tumors may have been too small torecruit therapeutic T cells from the periphery, a comparison was made ofi.v. versus i.c. delivery against larger day 19 PBT030-2 EGFP:ffLuctumors. For these studies, PBT030-2 engrafted mice were treated witheither two i.v. infusions (5×10⁶ CAR+ Tcm; days 19 and 26) or four i.c.infusions (1×10⁶ CAR+ Tcm; days 19, 22, 26 and 29) of IL13(EQ)BBZ+ Tcm,or mock Tcm (no CAR). Here too no therapeutic benefit as monitored byXenogen imaging or Kaplan-Meier survival analysis for i.v. administeredCAR+ T cells (FIGS. 12A and 12B). In contrast, potent antitumor activitywas observed for i.c. administered IL13(EQ)BBζ+ Tcm (FIGS. 12A-B). Next,brains from a cohort of mice 7 days post T cell injection were harvestedand evaluated for CD3+ human T cells by IHC. Surprisingly, for micetreated i.v. with either mock Tcm or IL13(EQ)BBζ Tcm there were nodetectable CD3+ human T cells in the tumor or in others mouse brainregions where human T cells typically reside (i.e. the leptomeninges)(FIG. 12C), suggesting a deficit in tumor tropism. This is in contrastto the significant number of T cells detected in the i.c. treated mice(FIG. 12D).

Tumor derived cytokines, particularly MCP-1/CCL2, are important inrecruiting T cells to the tumor. Thus, PBT030-2 tumor cells wereevaluated and it was found that this line produces high levels ofMCP-1/CCL2 comparable to U251T cells (data not shown), a glioma linepreviously shown to attract i.v. administered effector CD8+ T cells toi.c. engrafted tumors. Malignant gliomas are highly invasive tumors andare often multi-focal in presentation. The studies described aboveestablish that IL13BBZ T_(CM) can eliminate infiltrated tumors such asPBT030-2, and mediate long-term durable antitumor activity. The capacityof intracranially delivered CAR T cells to traffic to multifocal diseasewas also examined. For this study PBT030-2 EGFP:ffLuc TSs were implantedin both the left and right hemispheres (FIG. 13A) and CAR+ T cells wereinjected only at the right tumor site. Encouragingly, for all miceevaluated (n=3) we detected T cells by CD3 IHC 7-days post T cellinfusion both at the site of injection (i.e. right tumor), as wellwithin the tumor on the left hemisphere (FIG. 13B). These findingsprovide evidence that CAR+ T cells are able to traffic to and infiltratetumor foci at distant sites. Similar findings were also observed in asecond tumor model using the U251T glioma cell line (data not shown).

EXAMPLE 10 Comparison of Costimulatory Domains

A series of studies were conducted to evaluate various costimulatorydomains. The various CAR evaluated are depicted schematically in FIG.14A and included a first generation CD3ζ CAR lacking a costimulatorydomain, two second generation CARs incorporating either a 4-1BBcostimulatory domain or a CD28 costimulatory domain, and a thirdgeneration CAR containing both a CD28 costimulatory domain and 41BBcostimulatory domain. All CAR constructs also contain the T2A ribosomalskip sequence and a truncated CD19 (CD19t) sequence as a marker fortransduced cells.

CD4 and CD8 T_(CM) were lentivirally transduced and CAR-expressing Tcells were immunomagnetically enriched via anti-CD19. CD19 and IL13(i.e., CAR) expression levels as measured by flow cytometry. The resultsare shown in FIG. 14B. Stability of each CAR construct was determined bydividing the CAR (IL 13) mean flourescence intenstity (MFI) by that ofthe transduction marker (CD19t) (FIG. 14C). The two CAR including a4-1BB costimulatory domain exhibited the lowest expression levels ascompared to the CD19t transduction marker.

The ability of the indicated mock-transduced or CAR-expressing T cellsto kill IL13Rα2-expressing PBT030-2 tumor cell targets was determined ina 4-hour ⁵¹Cr-release assay at the indicated effector:target ratios. Theresults of this study are in FIG. 15A (mean % chromium release±S.D. oftriplicate wells are depicted). As expected, mock-transduced T cells didnot efficiently lyse the targets. In contrast, all CAR-expressing Tcells lysed the tumor cells in a similar manner. FIG. 15B depicts theresults of a study in which the indicated mock-transduced orCAR-expressing T cells were co-cultured overnight withIL13Rα2-expressing PBT030-2 tumor cells at a 10:1 ratio and supernatantswere analyzed for IL-13 and IFN-γ levels by cytometric bead array.Interestingly, T cells expressing the zeta, 41BB-zeta or CD28-41BB-zetaCARs exhibited lower antigen-stimulated cytokine production than T cellsexpressing the CD28-zeta CAR.

The in vivo efficacy of the various CAR was examined as follows.Briefly, NSG mice received an intracranial injection of ffLuc+ PBT030-2tumor cells on day 0, and were randomized into 6 groups (n=9-10 mice pergroup) for i.c. treatment with either PBS (Tumor Only), mock-transducedT cells or T cells expressing the indicated IL13Rα2-specific CAR on day8. Quantitative bioluminescence imaging was then carried out to monitortumor growth over time. Bioluminescence images for representative micein each group (FIG. 16A). Flux levels for each mouse at Day 27 (FIG.16B). All groups treated with IL13Rα2-specific CAR T cells, except thosetreated with T cells expressing the CD28-CAR, showstatistically-significant reduction in tumor volume compared to micetreated with mock-transduced T cells (FIG. 16C).

EXAMPLE 11 Amino Acid Sequence of IL13(EQ)BBζ/CD19t

The complete amino acid sequence of IL13(EQ)BBζ/CD19t is depicted inFIGS. 17A-B. The entire sequence (SEQ ID NO:1) includes: a 22 amino acidGMCSF signal peptide (SEQ ID NO:2), a 112 amino acid IL-13 sequence (SEQID NO:3; amino acid substitution E13Y shown in bold); a 229 amino acidIgG4 sequence (SEQ ID NO:4; with amino acid substitutions L235E andN297Q shown in bold); a 22 amino acid CD4 transmembrane sequence (SEQ IDNO:5); a 42 amino acid 4-1BB sequence (SEQ ID NO:6); a 3 amino acid Glylinker; a 112 amino acid CD3t sequence (SEQ ID NO:7); a 24 amino acidT2A sequence (SEQ ID NO:8); and a 323 amino acid CD19t sequence (SEQ IDNO:9).

The mature chimeric antigen receptor sequence (SEQ ID NO:10) includes: a112 amino acid IL-13 sequence (SEQ ID NO:3; amino acid substitution E13Yshown in bold); a 229 amino acid IgG4 sequence (SEQ ID NO:4; with aminoacid substitutions L235E and N297Q shown in bold); at 22 amino acid CD4sequence (SEQ ID NO:5); a 42 amino acid 4-1BB sequence (SEQ ID NO:6); a3 amino acid Gly linker; and a 112 amino acid CD3ζ sequence (SEQ IDNO:7). Within this CAR sequence (SEQ ID NO:10) is theIL-13/IgG4/CD4t/41-BB sequence (SEQ ID NO:11), which includes: a 112amino acid IL-13 sequence (SEQ ID NO:3; amino acid substitution E13Yshown in bold); a 229 amino acid IgG4 sequence (SEQ ID NO:4; with aminoacid substitutions L235E and N297Q shown in bold); at 22 amino acid CD4sequence (SEQ ID NO:5); and a 42 amino acid 4-1BB sequence (SEQ IDNO:6). The IL13/IgG4/CD4t/4-1BB sequence (SEQ ID NO:11) can be joined tothe 112 amino acid CD3ζ sequence (SEQ ID NO:7) by a linker such as a GlyGly Gly linker. The CAR sequence (SEQ ID NO:10) can be preceded by a 22amino acid GMCSF signal peptide (SEQ ID NO:2).

FIGS. 18A-O depict a comparison of the sequences ofIL13(EQ)41BBζ[L13{EQ}41BBζ T2A-CD19t_epHIV7; pF02630] (SEQ ID NO:12) andCD19Rop_epHIV7 (pJ01683) (SEQ ID NO:13).

EXAMPLE 12 Amino Acid Sequence of IL13(EQ)BBζ/CD19t

FIGS. 19-26 depict the amino acid sequences of additional CAR directedagainst IL13Rα2 in each case the various domains are labelled except forthe GlyGlyGly spacer located between certain intracellular domains. Eachincludes human IL13 with and Glu to Tyr (SEQ ID NO:3; amino acidsubstitution E13Y shown in highlighted). In the expression vector usedto express these CAR, the amino acid sequence expressed can include a 24amino acid T2A sequence (SEQ ID NO:8); and a 323 amino acid CD19tsequence (SEQ ID NO:9) to permit coordinated expression of a truncatedCD19 sequence on the surface of CAR-expressing cells.

A panel of CAR comprising human IL13(E13Y) domain, a CD28 tm domain, aCD28gg costimulatory domain, a 4-1BB costimulatory domain, and a CD3ζdomain CAR backbone and including either a HL (22 amino acids) spacer, aCD8 hinge (48 amino acids) spacer, IgG4-HL-CH3 (129 amino acids) spaceror a IgG4(EQ) (229 amino acids) spacer were tested for their ability tomediate IL13Ra2-specific killing as evaluated in a 72-hour co-cultureassay. With the exception of HL (22 amino acids) which appeared to havepoor CAR expression in this system, all were active.

1.-28. (canceled)
 29. A nucleic acid molecule encoding a chimericantigen receptor, wherein the chimeric antigen receptor comprises: ahuman IL-13 variant comprising the amino acid sequence of SEQ ID NO: 3with up to 5 single amino acid substitutions, provided that the aminoacid at position 11 of SEQ ID NO: 3 other than E; a transmembrane domainselected from: a CD4 transmembrane domain or variant thereof having 1-5amino acid substations, a CD8 transmembrane domain or variant thereofhaving 1-5 amino acid substitutions, a CD28 transmembrane domain or avariant thereof having 1-5 amino acid substations, and a CD3ζtransmembrane domain or a variant thereof having 1-5 amino acidsubstitutions; a costimulatory domain selected from the group consistingof: a CD28 costimulatory domain or a variant thereof having 1-5 aminoacid substitutions, a 4-1BB costimulatory domain or a variant thereofhaving 1-5 amino acid substitutions, and an OX40 costimulatory domain ora variant thereof having 1-5 amino acid substitutions; and CD3ζsignaling domain of a variant thereof having 1-5 amino acidsubstitutions.
 30. The nucleic acid molecule of claim 29, wherein thehuman IL-13 variant comprises the amino acid sequence of SEQ ID NO: 3.31. The nucleic acid molecule of claim 29, wherein the costimulatorydomain is a 4-1BB costimulatory domain or a variant thereof having 1-5amino acid substitutions.
 32. The nucleic acid molecule of claim 31,wherein the 4-1BB costimulatory domain comprises the amino acid sequenceof SEQ ID NO:
 6. 33. The nucleic acid molecule of claim 29, comprising aspacer region located between the IL-13 or variant thereof and thetransmembrane domain.
 34. The nucleic acid molecule of claim 33, whereinthe spacer comprises an IgG hinge region.
 35. The nucleic acid moleculeof claim 33, wherein the spacer region comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 4, 14-20, 50 and 51-52.36. The nucleic acid molecule of claim 29, wherein the CD3ζ signalingdomain comprises the amino acid sequence of SEQ ID NO:
 7. 37. Thenucleic acid molecule of claim 29, wherein a linker of 3 to 15 aminoacids is located between the costimulatory domain and the CD3ζ signalingdomain or variant thereof.
 38. The nucleic acid molecule of claim 37,wherein the linker comprises at least amino acids Gly Gly Gly.
 39. Thenucleic acid molecule of claim 29, wherein the nucleic acid moleculeexpresses a polypeptide comprising an amino acid sequence selected fromSEQ ID NOs: 10, 31-48 and
 52. 40. The nucleic acid molecule of claim 39,wherein the nucleic acid molecule expresses a polypeptide comprising SEQID NO:
 10. 41. The nucleic acid molecule of claim 29, wherein thechimeric antigen receptor comprises a IL-13/IgG4/CD4t/4-1BB regioncomprising the amino acid of SEQ ID NO: 11 and a CD3ζ signaling domaincomprising the amino acid sequence of SEQ ID NO:
 7. 42. The nucleic acidmolecule of claim 29, wherein the chimeric antigen receptor comprisesthe amino acid sequence of SEQ ID NOs: 10, 31-48 and
 52. 43. Apopulation of human T cells transduced by a vector comprising anexpression cassette encoding a chimeric antigen receptor, whereinchimeric antigen receptor comprises: a human IL-13 variant comprisingthe amino acid sequence of SEQ ID NO: 3 with up to 5 single amino acidsubstitutions, provided that the amino acid at position 11 of SEQ ID NO:3 other than E; a transmembrane domain selected from: a CD4transmembrane domain or variant thereof having 1-5 amino acidsubstations, a CD8 transmembrane domain or variant thereof having 1-5amino acid substitutions, a CD28 transmembrane domain or a variantthereof having 1-5 amino acid substations, and a CD3ζ transmembranedomain or a variant thereof having 1-5 amino acid substitutions; acostimulatory domain selected from the group consisting of: a CD28costimulatory domain or a variant thereof having 1-5 amino acidsubstitutions, a 4IBB costimulatory domain or a variant thereof having1-5 amino acid substitutions and an OX40 costimulatory domain or avariant thereof having 1-5 amino acid substitutions; and CD3ζ signalingdomain of a variant thereof having 1-5 amino acid substitutions.
 44. Thepopulation of human T cells of claim 43, wherein the chimeric antigenreceptor comprising an amino acid sequence selected from SEQ ID NOs: 10,31-48 and
 52. 45. The population of human T cells of claim 43, whereinthe chimeric antigen receptor comprises SEQ ID NO: 10.